ASSESSMENT OF ENDOCRINE ACTIVE SUBSTANCES USINGIN SILICO AND REPORTER ASSAYS IN CELL AND MICE
Tesi di Dottorato
Data di Pubblicazione:
2018
Citazione:
ASSESSMENT OF ENDOCRINE ACTIVE SUBSTANCES USINGIN SILICO AND REPORTER ASSAYS IN CELL AND MICE / P. La Fauci ; supervisor: M. Marinovich ; coordinatore: A. L. Catapano. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2018 Dec 17. 31. ciclo, Anno Accademico 2018. [10.13130/la-fauci-pierpaolo_phd2018-12-17].
Abstract:
In the last two decades, there have been growing scientific concern, public debate, and media attention over the possible deleterious effects in humans and wildlife that may result from exposure to substances that have the potential to interfere with the endocrine system. Endocrine disrupting compounds (EDCs) encompass a variety of substance classes, including natural and synthetic hormones, plant constituents, pesticides, substances used in industry and in consumer products, pollutant. It is well documented that EDCs targets are mainly the nuclear receptors (NRs) such as the sexual hormones estrogen receptors ERs. ER is involved in a broad spectrum of physiological processes in different organs and tissues as well as in several diseases, such as breast and endometrial cancer, osteoporosis, and prostate hypertrophy, neurodegenerative diseases or in immune system activation. The regulatory agencies for the protection of human health and wildlife have been issuing the necessity to investigate and clarify the mode of action of these exogenous substances by the development of alternative toxicological methods such as in silico model and in vitro testing in order to predict the toxicity of these EDCs. This research had the ambitious aim to develop an integrated toxicological strategy based on the combination of available information of endocrine disrupting activity retrieved from the scientific literature, in silico model, imaging methodologies applied to reporter systems in vitro and in vivo and ex vivo to predict among a set of chemicals those with an endocrine disrupting activity or ability to activate other toxicological pathways such as inflammation and oxidative stress measured in the male reproductive organs and in the genital and abdominal area of mice. The selected molecules range from known (DES) to suspected (BPA) endocrine disruptors and included both synthetic (DEHP) and natural (genistein) compounds. The first step used was in silico analysis with evaluation of the possible binding of selected substances to the estrogen alpha receptor to support the hypothesis that their hormonal activity occurred through a receptorial mechanism. This approach is commonly used and is also part of the first level of investigation suggested by EFSA/ECHA in the recognition of EDCs. The computational methodology estimated different values of affinity of each ligand to hER Ligand Binding Domain (LBD). The use of two different approaches (XP GLIDE SCORE and MMGBSA dG Bind) also allowed for solvation to be taken into account. That meant that MMGBSA protocol considered both the interactions of some water molecules with the LBD and the solvent-ligand ones. The estradiol showed the best affinity values in both approaches as being the endogenous hormone was able to contrast the solvation effect. The dissociation constant (Ki) values calculated from the XP GLIDE SCORE fitted well with the Ki experimentally determined in vitro binding assay by other research groups. Thus, the computed Ki has been chosen as parameter for the prediction of putative endocrine disruptor activity. However, the lack of correlation between the (computed and experimental) Ki and in vivo experimental observed ED activity (from literature data) for all chemicals (only estradiol and zearalenone have similar affinity to ER and literature evidences of ED activity; genistein has a good Ki values but not ED activity such as BPA and methoxychlor), did not allow a prioritization of the investigated chemicals for ED activity through the results of their affinity. In the next step, to check if the receptor binding well correlated with the hormonal activity, the ER Reporter gene assay was performed, based on the ability of a compound to stimulate ER-dependent transcriptional activity in
Tipologia IRIS:
Tesi di dottorato
Keywords:
endocrine system; endocrine disrupting compounds; estrogen receptor; assessment; in silico method; in vitro bioluminescence assay; in vivo bioluminescence experiments; dynamic; non-invasice; classification; EDC; threshold; BPA; DES; GENISTEIN; ZEARALENONE; METHOXYCHLOR; VINCLOZOLIN; DEHP; scientific literature review
Elenco autori:
P. LA FAUCI
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